Combination therapy using ribavirin as elF4E inhibitor

ABSTRACT

The present invention relates to pharmaceutical compositions and combination therapies for treating patients having a neoplasm or a proliferative disorder, the combination comprises ribavirin, GDC-0449 and a chemotherapeutic agent, wherein said combination therapy overcomes resistance developed in patients during anti-neoplastic treatment. The present invention also provides a combination therapy for treating patients having leukemia.

RELATED APPLICATION DATA

This application is a continuation of U.S. patent application Ser. No.14/344,536, filed Mar. 12, 2014, now U.S. Pat. No. 9,545,416, whichapplication is a 371 application of International Application No.PCT/CA2012/000831, filed Sep. 13, 2012, and which application claimspriority to CA Patent Application No. 2,752,008, filed Sep. 13, 2011.Each of these applications is incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

The present invention relates to use of combination therapy for treatingpatients having a neoplasm, a proliferative disorder, a pre-neoplasm ora precancerous lesion, comprising administering to a patient atherapeutically effective amount of an inhibitor of eukaryotictranslation initiation factor 4E (eIF4E) and a therapeutically effectiveamount of a chemotherapeutic agent, wherein said combination treatmentovercomes resistance developed in patients during anti-neoplastictreatment.

BACKGROUND OF THE INVENTION

Cancer, tumor-related disorders, and neoplastic disease states areserious and oftentimes life-threatening conditions. These diseases anddisorders, which are characterized by rapidly-proliferating cell growth,continue to be the subject of research efforts directed toward theidentification of therapeutic agents which are effective in thetreatment thereof. Such agents prolong the survival of the patient,inhibit the rapidly-proliferating cell growth associated with theneoplasm, or effect a regression of the neoplasm. One class of cancer isleukemia which consists of malignancies derived from hematopoietic(blood-forming) cells. Part of this class of cancers is acute myeloidleukemia (AML), also known as acute myelogenous leukemia, which is acancer of the myeloid line of blood cells, characterized by the rapidgrowth of abnormal white blood cells that accumulate in the bone marrowand interfere with the production of normal blood cells. AML is the mostcommon acute leukemia affecting adults, and its incidence increases withage.

In order to treat patients diagnosed with cancer, scientific researchersaround the world have investigated a multitude of mutant cancer cells,genetic mutations, site-specific mutagenesis, DNA, RNA, RNA and proteinexpression, transporters, genetic sequencing, so as to map biochemicalpathways in cancer cells at the molecular level and find the “cure” tovarious types of cancer and/or the ability to manage these as chronicdiseases. One of the more recent cancer research fields consists of theinvestigation of the deregulation of the RNA metabolism that contributesto cells becoming cancerous, and even more specifically, the inhibitionof a specific factor, eukaryotic translation initiation factor 4E(eIF4E), by a well-known anti-viral drug, ribavirin, which impedeseIF4E's ability to make cells cancerous without significantly affectingnormal cells.

Ribavirin is chemically designated as:1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-1H-1,2,4-triazole-3-carboxamide, and has the following chemicalstructure:

The preparation of ribavirin is disclosed in U.S. Pat. No. 3,798,209.The clinical pharmacology of ribavirin is also disclosed in Glue, “Theclinical pharmacology of ribavirin” Seminars in Liver Disease, vol. 19,suppl. 1, 1999, p. 17-24, 1999.

Canadian Patent Nos. 2,287,056 and 2,323,849 disclose an orallyadministrable solid dosage form containing a compacted ribavirincomposition as well as a process for making such solid dosage forms.

A further discussion on the interaction of ribavirin with eIF4E can befound in: Assouline et al., “Molecular targeting of the oncogene eIF4Ein acute myeloid leukemia (AML): a proof-of-principle clinical trialwith ribavirin” Blood, Vol. 114, no. 2 (2009); Borden, “Tissue Targetingin Cancer: eIF4E's Tale” Clin. Cancer Res., 2009); Borden et al.,“Ribavirin targets eIF4E dependent Akt survival signalling”, Biochem.Biophys. Res. Commun., Vol. 375(3): 341-345 (Oct. 24, 2008); Kraljacic,et al., “Inhibition of eIF4E with ribavirin cooperates with commonchemotherapies in primary acute myeloid leukemia specimens” Leukemia 25,1197-1200 (July 2011) and other references known in prior art.

Canadian Patent Application No. 2,685,520 discloses compounds that areuseful in treating viral infections and cancer, pharmaceuticalcompositions comprising the compounds, and synthetic methods andintermediates that are useful for preparing the compounds. The compoundsthat are useful as anti-viral agents and/or anti-cancer agents includeribavirin.

Canadian Patent Application No. 2,715,885 discloses novel compoundsprovided for use in the treatment of tumors and the prophylaxis ortreatment of viral infections, wherein one of the anti-viral agents isribavirin.

Canadian Patent Application No. 2,674,589 discloses compounds, as wellas pharmaceutical compositions comprising the compounds that are usefulas anti-viral agents and/or as anti-cancer agents, wherein the one ofthe anti-viral agents is ribavirin.

Canadian Patent Application No. 2,430,966 discloses anilinopyrimidinederivatives as JNK pathway inhibitors and compositions comprisingadministering an effective amount of an anti-cancer agent, wherein oneof the proposed anti-cancer agents is ribavirin or cytarabine.

The eukaryotic translation factor, eIF4E, is found in all cells and isimportant to make new proteins. In cancer patients, the amount of eIF4Eis overexpressed in AML, and is abnormally high in 30% of cancers,including the particularly aggressive subtypes of myeloid leukemiareferred to as M4 and M5. The function of eIF4E to make new proteinsdepends upon its ability to bind to the front part of RNA known as them⁷G cap (7-methyl guanosine) (located at the 5′ end of the mRNA), whichthen allows the cell to “translate” or turn this RNA into protein. Italso has a role in the export of the mRNA into the cytoplasm, which mustprecede the translation step. It is known in the art that cancer cellswith elevated levels of eIF4E seem to have developed an oncogeneaddiction to eIF4E.

Examples and reference can be seen in the prior art as follows:International laid-open publication nos. WO 2007/123579 and WO2008/060369 (Translational Therapeutics); International laid-openpublication no. 2010/006291 (Nodality Inc.), and U.S. Pat. Nos.7,425,544 and 7,601,700 and International laid-open publication no. WO2005/028628 (Eli Lilly and Co. and ISIS Pharmaceuticals Inc.), as wellas Canadian Patent Application No. 2,632,903 (Nabil-Habib Lab andVianova Labs Inc.) and some others.

Thus, because of its properties, the eukaryotic translation factor,eIF4E, has therefore become an appealing clinical target to treatpatients diagnosed with cancer, in particular AML. In this connection,targeting of the eIF4E-m⁷G cap-binding activity has been studied in aphase II trial, in leukemia patients, and has been reported in Assoulineet al., “Molecular targeting of the oncogene eIF4E in acute myeloidleukemia (AML): a proof-of-principle clinical trial with ribavirin”Blood, Vol. 114, No. 2 (Jul. 9, 2009, Epub 2009 May 11). In this trial,the commonly used anti-viral drug, ribavirin, was found to decrease thefunction of eIF4E because it mimics the m⁷G cap; thus inhibitingeIF4E-induced export and translation of sensitive transcripts. In cellculture experiments, ribavirin did not modulate the levels of eIF4Eprotein or RNA. However in patients, ribavirin not only inhibits eIF4E,it also can lead to the downregulation of eIF4E protein (and RNA) levelsas observed in patients in a phase II clinical trial using ribavirinmonotherapy. Finally, in living cells, it was demonstrated that eIF4Ebinds ³H ribavirin further supporting the idea that eIF4E bindsribavirin directly in vitro and in vivo.

Several advantages have been disclosed in the prior art and from thesedisclosures, it can be understood that the physical mimicking of thenatural ligand of eIF4E, ribavirin, preferentially inhibits the growthof primary AML (M4/M5 AML) specimens with elevated eIF4E levels relativeto specimens with normal levels of eIF4E (e.g., M1/M2 AML) or normalcontrols. It is also taught that when ribavirin monotherapy is used, notreatment-related toxicities are observed. Further studies indicate that³H ribavirin immunoprecipitates (IPs) with eIF4E in living cells furthersupport the claim that ribavirin directly binds eIF4E.

In conducting clinical trial no. NCT00559091, the Applicant observedthat many patients had resistance prior to the start of ribavirintherapy due to the other therapies they received or de novo. Also allresponding patients became resistant to ribavirin monotherapy. In somepatients, monotherapy had no impact suggesting that they were resistantprior to the start of treatment. Thus, a problem associated with aribavirin monotherapy for use in cancer treatment, is that AML cellsbecome resistant prior to the start of ribavirin therapy due to theother therapies or become resistant as a result of ribavirin treatment(primary versus acquired resistance, respectively).

In fact, leukemic cells become resistant to nearly all monotherapieswithin two (2) to four (4) months. To overcome this issue of resistance,it is not uncommon in the clinical field, and as for most treatmentsinvolving monotherapy, to combine such treatment, simultaneously orsequentially, with chemotherapy. The use of chemotherapeutic agents hasmany secondary effects on patients, including and not limited to damageof normal cells, anemia, bleeding, constipation, fatigue, hair loss,infections, memory changes, swelling, and even death, amongst manyothers. Conventional chemotherapy also requires a stay at the hospitalso as to administer the chemotherapeutic agent(s) as well as supportivecare for the side effects.

One of chemotherapeutic agents known for treating cancer is cytarabine,also known as Ara-C® (arabinofuranosyf cytidine or cytosinearabinoside), which is chemically designated as4-amino-1-[(2R,3S,4R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]pyrimidin-2-one. It has the following chemical formula:

Cytosine arabinoside is a chemotherapy antimetabolic agent used mainlyin the treatment of cancers of white blood cells such as acute myeloidleukemia (AML) and non-Hodgkin lymphoma. It destroys cancer cells byinterfering with DNA synthesis. Its mode of action is due to its rapidconversion into cytosine arabinoside triphosphate, which damages DNAwhen the cell cycle holds in the S phase (synthesis of DNA). Rapidlydividing cells, which require DNA replication for mitosis, are thereforemost affected. Cytosine arabinoside also inhibits both DNA and RNApolymerases and nucleotide reductase enzymes needed for DNA synthesis.

Cytosine arabinoside combines a cytosine base with an arabinose sugar.Cytosine normally combines with a different sugar, deoxyribose, to formdeoxycytidine, a component of DNA. Certain sponges, where it wasoriginally found, use arabinoside sugars to form a different compound(not part of DNA). Cytosine arabinoside is similar enough to humancytosine deoxyribose (deoxycytidine) to be incorporated into human DNA,but different enough that it kills the cell. This mechanism is used tokill cancer cells. Cytarabine is the first of a series of cancer drugsthat altered the sugar component of nucleosides.

Many combinations of drugs have been developed to treat patientsdiagnosed with cancer, including, for example, AML. For example, Zhu etal., “Novel agents and regime for acute myeloid leukemia: 2009 ASHannual meeting highlights” Journal of Hematology & Oncology 2010, 3:17(Review) discloses monotherapies of daunorubicin, voreloxin, ARRY-520,AZD1152, AZD6244 and terameprocol, as well as combinations of drugs suchas: cytarabine with daunorubicin; fludarabine, cytarabine withidarubicin, mitoxantrone with cytarabine; clofarabine alone or incombination with low-dose Ara-C® or high dose Ara-C® with the monoclonalantibody GO; combination therapy with sorafenib; tipifarnib withbortezomib; azacitidine with botezomib or low-dose GO; amonafile withAra-C®; lenalidomine, Ara-C® and daunorubicin; as well as ribavirin withAra-C®, in the treatment of elderly AML or relapsed AML or refractoryAML.

The combination of ribavirin and low-dose Ara-C®, Ara-C® and idarubicin,and combinations thereof (i.e. ribavirin, Ara-C® and idarubicin) as wellas sorafenib with ribavirin was specifically disclosed by Assouline etal. in “Targeting the oncogene eIF4E with ribavirin: a novel therapeuticavenue in acute myeloid leukemia” Blood 114, 2009 and by Kraljacic etal. in “Inhibition of eIF4E with ribavirin cooperates with commonchemotherapies in primary acute myeloid leukemia specimens” Leukemia 25,(2011).

Clinical trials have also been conducted on several combinations ofdrugs for the treatment of leukemia and/or AML, and are available at:clinical trials.gov (NTC01056523) andhttp://clinicaltrials.gov/ct2/home.

Examples of combinations of therapy for AML, include and are not limitedto: ABT-348; ABT-888 and topotecan with or without carboplatin;alemtuzumab, busulfan, and cyclophosphamide; alemtuzumab, busulfan, andmelphalan; alemtuzumab with fludarabine phosphate; all-trans retinoicacid with bryostatin 1; amifostine trihydrate, cytarabine withmitoxantrone hydrochloride; arsenic trioxide; azacitidine withcytarabine (also referred to as Ara-C (Registered trademark));azacitidine, asparaginase, cytarabine, aunorubicin hydrochloride,etoposide, lintuzumab with thioguanine; azacitidine with arsenictrioxide; azacitidine with belinostat; azacitidine with entinostat;azacitidine with gemtuzumab ozogamicin; azacitidine with lenalidomide;azacitidine with midostaurin; 5-azacytidine (Vidaza (Registeredtrademark)) with panobinostat (lbh589); 5-azacytidine (5-aza), valproicacid with all-trans retinoic acid (atra); azacytidine with valproicacid; azacitidine with phenyl butyrate; basiliximab; becatecarin;belinostat; bendamustine; bevacizumab, cytarabine with mitoxantronehydrochloride; bexarotene and gm-csf; BMS-214662; bortezomib withbelinostat; bortezomib with melphalan; bortezomib and vorinostat;bryostatin 1; busulfan, filgrastim with etoposide; busulfan withfludarabine; busulfan, cyclophosphamide, mycophenolate mofetil withtacrolimus; carboplatin, docetaxel with ifosfamide; cediranib maleate;clofarabine; clofarabine with cyclophosphamide; clofarabine, cytarabinewith idarubicin; clofarabine, filgrastim with cytarabine; clofarabineand high-dose melphalan; clofarabine, melphalan, and thiotepa;cilengitide; cixutumumab with temsirolimus; CPX-151; CT53518; cytarabineand daunorubicin with or without gemtuzumab ozogamicin; cytarabine anddaunorubicin with or without zosuquidar trihydrochloride; cytarabine,idarubicin with tipifarnib; cytarabine with 7-hydroxystaurosporine;cytarabine with laromustine; cytarabine with tanespimycin; cytarabinewith triapine; cyclophosphamide; cyclosporine and Given IV withmycophenolate mofetil; cyclosporine, mycophenolate mofetil, andpentostatin; cyclosporine, methotrexate, methoxsalen, mycophenolatemofetil with pentostatin; decitabine; decitabine with lenalidomide;decitabine with romidepsin; decitabine with tretinoin; decitabine withvalproic acid; decitabine with vorinostat (sequential); deferasirox;dolastatin; eltrombopag olamine; entinostat; everolimus; exatecanmesylate; fentanyl citrate; flavopiridol and vorinostat; fludarabine andcyclophosphamide as well as total-body irradiation, followed bycyclosporine and mycophenolate mofetil; fludarabine phosphate with GivenIV; fludarabine phosphate with tretinoin; fludarabine, carboplatin, andtopotecan; fludarabine, carboplatin, topotecan with thalidomide;fludarabine with melphalan; fludarabine with thiotepa; fludarabine withtreosulfan; gimatecan; 7-hydroxystaurosporine with perifosine;hydroxyurea with laromustine; idarubicin with saha (vorinostat);ipilimumab; imatinib mesylate; interleukin-12 followed by interferonalfa; irofulven; itraconazole with midostaurin; ispinesib; JNJ-26481585;KW-2449; laromustine; lintuzumab; lonafarnib; MEK inhibitor AZD6244;MS-275 and gm-csf; MGCD0103; MLN8237; mycophenolate mofetil, tacrolimuswith daclizumab; ON 0191 O.na; OX14503; palivizumab with or withoutribavirin; paricalcitol; phenyl butyrate and tretinoin; procrit;pyroxamide; fluorouracil, leucovorin calcium, and topotecanhydrochloride; rasburicase; revlimid; romidepsin; sargramostim,amifostine trihydrate, carboplatin with cyclophosphamide; S81518;SJG-136; STA-9090; sirolimus with tacrolimus; sodium salicylate;sorafenib tosylate; sorafenib with vorinostat; tacrolimus andmycophenolate mofetil with or without sirolimus; tacrolimus andmycophenolate mofetil; tetradecanoylphorbol acetate; temsirolimus;tipifarnib; triapine with fludarabine phosphate; vorinostat; and yttriumy 90 anti-cd45 monoclonal antibody ahn-12, amongst others.

As noted in the prior art, the population having advanced AML haddifficulty receiving more than one cycle of therapy. Anti-leukemiaactivity could be observed with relapsed/refractory disease. Anotherproblem associated with AML drug therapies is epigenetic silencing; aphenomenon by which a drug-induced increased methylation allows foracquired drug resistance. The contribution of epigenetic mechanisms forcorrect cell function is highlighted by the effects of theirderegulation that, in cooperation with genetic alterations, lead to theestablishment and progression of tumors (see Fazi et al., in“Heterochromatic gene repression of the retinoic acid pathway in acutemyeloid leukemia”, Blood, May 2007, vol. 109(10), p. 4432-4440).

Other problems with concomitant drug therapy is that the drugs mayproduce antagonistic effects, undergo collateral sensitivity/resistanceto other drugs, be difficult to determine the right dosing regimen, havetoxicity issues; and create multiple drug resistance. From the above, itbecomes apparent that the treatment of myelodyplastic syndromes (MDS)and/or AML remains a challenge to the clinician despite recent advances.Many patients either will not respond or will have only limited and/orbrief responses to single agent therapy or even concomitant therapy.

Even in the early stage of clinical trials, some side effects have beenobserved, which were due to low dose Ara-C®. Hemolysis has also beenobserved in a patient treated with the combination of ribavirin andAra-C®. This phenomenon can be attributed to ribavirin, but such sideeffects were not observed in the ribavirin monotherapy trial (NCTNCT00559091). It is possible that Ara-C® somehow potentiates this sideeffect. No therapy related side effects were observed with ribavirinalone.

Most virus studies have primarily focused on the effects of ribavirin onthe virus, for example: mutations in viral polymerases, which is not thecase in the context of the present invention. In the viral context,ribavirin impedes growth of the virus and resistance occurs when thevirus continues to replicate even in the presence of ribavirin. In thecancer context, it is a measure of cells becoming resistant to theanti-proliferative effects of ribavirin, i.e. that eIF4E mediatesproliferation, ribavirin impedes this effect and then eventually, thecells continue to proliferate even in the presence of ribavirin.Further, there could be different biochemical pathways modulated. Thus,one cannot compare viral infections, such as the hepatitis C virus (HCV)or the poliovirus, with cancerous type cells or cell growth as themechanisms of action and resistance are completely different.

Drug resistance is a major impediment in cancer research, particularlyfor SCLC because of limited recent innovations in treatment methods. Onepossible explanation for chemoresistance is activation of the hedgehogsignaling pathway, which promotes cellular proliferation anddifferentiation and has been implicated in chemoresistance. Its geneexpression was examined in resistant SCLC cell lines and reportedaberrant expression of hedgehog pathway-related genes, among which GUIwas particularly significant. GLI1 is a transcription factor involved incell fate determination, proliferation, oncogenesis, and cancerprogression.

With the use of new effective chemotherapy, hormone therapy, andbiological agents and with information regarding more effective ways tointegrate systemic therapy, surgery, and radiation therapy, elaboratingan appropriate treatment plan is becoming more complex. To offer bettertreatment with increased efficacy and low toxicity, selecting therapiesbased on the patient and the clinical and molecular characteristics ofthe tumor is necessary.

Therefore, accordingly a need exists to overcome the aforementioneddrawbacks by a combination therapy.

SUMMARY OF THE INVENTION

It has been found that a particular combination of ribavirin (RBV) witha therapeutically effective amount of hedgehog pathway inhibitor andcytarabine (Ara-C (Registered trademark)), or some otherchemotherapeutical agent, overcomes, for the most part, theaforementioned drawbacks.

In one aspect of the present invention there is provided apharmaceutical composition suitable for use in treating a neoplasm or aproliferative disorder, wherein the composition comprises an inhibitorof eIF4E, a hedgehog pathway inhibitor, and a pharmaceuticallyacceptable carrier. In a further embodiment, there is provided apharmaceutical composition suitable for use in treating a neoplasm or aproliferative disorder, wherein the composition comprises an inhibitorof eIF4E, a hedgehog pathway inhibitor, a chemotherapeutic agent and apharmaceutically acceptable carrier.

Another aspect of the present invention is directed to a use of apharmaceutical composition for treating a neoplasm or a proliferativedisorder, wherein the composition comprises an inhibitor of eIF4E, ahedgehog pathway inhibitor, and a pharmaceutically acceptable carrier.

In a further aspect of the present invention, there is provided a use ofan inhibitor of eIF4E, a hedgehog pathway inhibitor, and apharmaceutically acceptable carrier, for the manufacture of a medicamentfor the treatment of a neoplasm or a proliferative disorder.

An aspect of the present invention is directed to overcoming existingdisadvantages in treatment of neoplasm, a proliferative disorder, apre-neoplasm or a precancerous lesion by use of combination therapy,comprising an inhibitor of eIF4E and a chemotherapeutic agent toovercome resistance developed in patients during anti-neoplastictreatment.

Another aspect of the present invention is to provide a combinationtherapy for treating a neoplasm, a proliferative disorder, apre-neoplasm or a precancerous lesion, comprising administering to apatient a therapeutically effective amount of an inhibitor of eIF4E witha therapeutically effective amount of a hedgehog pathway inhibitor andwith or without a therapeutically effective amount of a chemotherapeuticagent, wherein said combination therapy overcomes resistance developedin patients during anti-neoplastic treatment.

An aspect of the present invention is directed to use of combinationtherapy for treating patients having a neoplasm or a proliferativedisorder, comprising an inhibitor of eIF4E and a chemotherapeutic agent,wherein said combination therapy overcomes resistance developed inpatients during anti-neoplastic treatment.

A further aspect of the present invention is directed to the use of thecombination therapy for treating a neoplasm, a proliferative disorder, apre-neoplasm or a precancerous lesion, comprising administering to apatient a therapeutically effective amount of an inhibitor of eIF4E, atherapeutically effective amount of a hedgehog pathway inhibitor with orwithout a therapeutically effective amount of a chemotherapeutic agent,wherein said combination therapy overcomes resistance developed inpatients during anti-neoplastic treatment.

Another aspect of the present invention is directed to the use ofcombination therapy for treating patients having a neoplasm or aproliferative disorder, comprising an inhibitor of eIF4E and achemotherapeutic agent, wherein said combination therapy minimizes orprevents the growth of resistant cells developed in patients duringanti-neoplastic treatment.

Another aspect of the present invention provides a pharmaceuticalcomposition for treating a neoplasm, a proliferative disorder, apre-neoplasm or a precancerous lesion, wherein the pharmaceuticalcomposition comprises an inhibitor of eIF4E, a hedgehog pathwayinhibitor and a chemotherapeutic agent. Preferably the inhibitor ofeIF4E is ribavirin, the inhibitor hedgehog pathway inhibitor is GDC-0449(but similar hedgehog pathway inhibitors may also be employed), and thechemotherapeutic agent is cytarabine (Ara-C (Registered trademark)).

Also preferably, the therapeutically effective amount of ribavirin andthe therapeutically effective amount of GDC-0449 are administratedsimultaneously or sequentially in resistant cells lines. More preferablythe GDC-0449 is treated at 3 nM for 2 days prior to start of ribavirintreatment.

Another aspect of the present invention provides a pharmaceuticalcomposition for treating a neoplasm, a proliferative disorder, apre-neoplasm or a precancerous lesion, wherein the therapeuticallyeffective amount of the inhibitor of the eIF4E gene product and thetherapeutically effective amount of the hedgehog pathway inhibitor areadministrated simultaneously or sequentially prior to initiatingadministration of the chemotherapeutic agent.

Yet another aspect of the present invention is directed to a use ofcombination therapy in treating the neoplasm or proliferative disorder,comprising administering to a patient a therapeutically effective amountof an inhibitor of eIF4E, a therapeutically effective amount of ahedgehog pathway inhibitor and a therapeutically effective amount of achemotherapeutic agent, wherein said combination therapy overcomesresistance developed in patients during anti-neoplastic treatment.

Preferably, the inhibitor of eIF4E is ribavirin, which is administeredin an amount between about 1000 to about 4400 mg per day. Alsopreferably, the chemotherapeutic agent is cytarabine (Ara-C (Registeredtrademark)) administered in a low dose, wherein the low dose ofcytarabine (Ara-C (Registered trademark)) ranges from about 5 mg/day toabout 20 mg twice a day. More preferably, the Ara-C (Registeredtrademark) dose ranges between about 10 mg/day to 20 mg twice a day.

Yet another aspect of the present invention is directed to a use of aninhibitor of eIF4E, in combination therapy with a chemotherapeutic agentto overcome resistance developed in patients during anti-neoplastictreatment.

Preferably, said combination therapy comprises administering to patienta therapeutically effective amount of ribavirin, simultaneously orsequentially with a therapeutically effective amount of the cytarabine(Ara-C (Registered trademark)).

Also preferably, said combination therapy comprises administering to thepatient the therapeutically effective amount of ribavirin, wherein theplasma levels of ribavirin range from 4-10 μM, as determined by massspectrometry. More preferably, the plasma levels of ribavirin are above20 μM, as determined by mass spectrometry.

A further aspect of the present invention is directed to a method of usefor the combination therapy for treating a neoplasm or proliferativedisorder, a pre-neoplasm or a precancerous lesion, wherein said methodcomprises administering to patient an inhibitor of the eIF4E geneproduct, simultaneously or sequentially with a therapeutically effectiveamount of a hedgehog pathway inhibitor and with or without thechemotherapeutic agent.

A further aspect of the present invention is directed to use ofcombination therapy for treating a neoplasm or a proliferative disorder,wherein the treatment comprises administration of ribavirin with achemotherapeutic agent, wherein said combination therapy reduces eIF4Elevels and re-localizes the eIF4E gene product.

Preferably, the combination therapy comprises administration of aninhibitor of eIF4E in combination with a chemotherapeutic agent, whereinsaid combination therapy provides a collateral sensitivity to a thirdactive agent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a Western analysis of CD34+ cells isolated from normal (norm),M5 AML or two blast crisis (bc) CML specimens. Actin is shown forloading. Results are representative of >100 primary specimens. It isshowed that eIF4E is substantially upregulated and forms abnormallylarge nuclear bodies in a subset of AML (FAB subtype M4/M5) and blastcrisis CML primary specimens. Further, eIF4E dependent mRNA export oftargets such as cyclin D1 is substantially upregulated leading toincreased protein levels for these targets.

FIG. 2 is a diagram demonstrating ENT1 and ADK RNA levels as a functionof ribavirin treatment in patients. CT is the number of 28 day cycles ofribavirin, normal is derived from normal CD34+ cells from the bonemarrow. There was no more before treatment RNA available for patient 11,but he was responding at cycles 3 and 4 and clinical and molecularrelapse occurred at cycle 9. For instance, in Patient 11 (CR), weobserved a 20-fold reduction in ENT1 and a 5-fold reduction in ADK RNAlevels at relapse relative to during response (after 9 cycles oftreatment).

FIG. 3 is a western blot of three primary multiple myeloma specimensversus CD34+ cells isolated from normal bone marrow. It was determinedthat eIF4E levels are elevated in 3/3 multiple myeloma (MM) (FIG. 3),and 2/2 MDS specimens. Only 1/8 B-ALL specimens had elevated eIF4E.However, the one specimen that had elevated eIF4E contained a t4; 11translocation.

FIG. 4 is a diagram showing Type II resistance: Normal uptake, loss ofeIF4E-ribavirin interaction. Fold enrichment of 14C ribavirin overnegative control (IP IgG).

FIG. 5 is a graphical demonstration of the uptake of 3H-ribavirin as apotential mechanism for ribavirin resistance.

FIG. 6 is a diagram demonstrating knocking down Gli-1 reverts resistanceand overexpressing Gli-1 imparts it. Aiming for 50% reduction forresponse as the experiment is done at the IC50 of this cell line.

FIG. 7 is a diagram demonstrating Gli-1 hedgehog pathway inhibitorGDC-0449 (Roche) partially reverting ribavirin resistance. One treatmentwith 3 nM GDC-0449 followed 48 hours later with ribavirin.

FIG. 8 is a Western analysis of glucuronidation pathway enzymes (UGT1As)which are elevated in F10R resistant cells with normal uptake relativeto parental cell lines or other resistant ones.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the field of pharmaceutics, andespecially to use of combination therapy for treating patients having aneoplasm, a proliferative disorder, a pre-neoplasm or a pre-cancerouslesion. In particular, the present invention is directed to thetreatment of neoplasm (i.e. cancers), proliferative disorders,pre-neoplasm and precancerous lesions, with a combination therapy,comprising an inhibitor of eIF4E, a hedgehog pathway inhibitor and achemotherapeutic agent, wherein said combination therapy overcomesprimary or acquired resistance developed in patients duringanti-neoplastic treatment.

Definitions

A “prodrug” is a pharmacological substance (drug) administered in aninactive (or significantly less active) form. Once administered, theprodrug is metabolised in vivo into an active metabolite, a processtermed bioactivation. The rationale behind the use of a prodrug isgenerally for absorption, distribution, metabolism, and excretion (ADME)optimization. Prodrugs are usually designed to improve oralbioavailability, with poor absorption from the gastrointestinal tractusually being the limiting factor. Additionally, the use of a prodrugstrategy increases the selectivity of the drug for its intended target.

An example of this can be seen in many chemotherapy treatments, in whichthe reduction of adverse effects is always of paramount importance.

The abbreviation “eIF4E” stands for eukaryotic translation initiationfactor 4E, which is a protein which in humans is encoded by the eIF4Egene product.

By an “inhibitor of eIF4E” as used herein, is meant any compound thatinhibits the biochemical activity of the eIF4E gene product includingits role in mRNA translation and mRNA export or eIF4E levels (RNA or 25protein). An example of inhibitors of eIF4E is ribavirin(1-13,0-ribofuranosyl-1H-1,2,4-thiazole-3-carboxamide) and itsderivatives. Preferably, an “inhibitor of eIF4E” results in a reductionin cancer or dissemination of, for example, at least 10%, 20%, 30%, 40%or 50%. In more preferable embodiments, an “inhibitor of eIF4E” reducesreplication or dissemination, for example, by at least 60%, 70%, 80%,90%, 95%, or even 99%, of cancer cells.

The term “GDC-0449” means the inhibitor of Hedgehog-Gli pathway which isdeveloped for potential use in cancer treatment. GDC-0449 is asmall-molecule inhibitor designed to specifically inhibit SMO, a keymediator of the Hh signaling pathway. GDC-0449 was discovered byGenentech and was jointly validated with Curis, Inc. through a series ofpreclinical studies.

The term “chemotherapeutic agent” as used herein, means a drug used intreatment of cancer usually an antineoplastic drug or with a combinationof such drugs into a standardized treatment regimen. Most chemotherapyagents and medications work by interfering with DNA synthesis orfunction. Based on their chemical action at a cellular level,chemotherapy agents can be classified as cell-cycle specific agents(effective during certain phases of cell cycle) and cell-cyclenonspecific agents (effective during all phases of cell cycle).Depending on their characteristics and nature of treatment, chemotherapyagents can be categorized as alkylating agents, antimetabolites,anthracyclines, antitumor antibiotics, monoclonal antibodies, platinums,or plant alkaloids.

The term “low dose” as referred to herein, means an amount of Ara-C® forrepressing the tumorigenicity of cells.

The terms “cancer”, “cancerous” or “neoplasm” or “neoplastic cells”comprises neoplasm, cancers, or neoplastic cells located at the originalsite of proliferation (“primary tumor or cancer”) and their invasion ofother tissues, or organs. They also refer to or describe thephysiological condition in mammals in which a population of cells ischaracterized by unregulated cell growth.

Examples of cancer include and are not limited to: leukemia, acutemyeloid leukemia, acute myelocytic leukemia, acute myeloblasticleukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia,acute monocytic leukemia, acute erythroleukemia, chronic leukemia,chronic myelocytic leukemia, chronic lymphocytic leukemia, polycythemiavera, lymphoma, Hodgkin's disease, non-Hodgkin's disease lymphoma,Waldenstrom's macroglobulinemia, heavy chain disease, fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,angiosarcoma, endotheliosarcoma, lyinphangiosarcoma, synovioma,mesothelioma, lymphangioendotheliosarcoma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterinecancer, testicular cancer, lung carcinoma, small cell lung carcinoma(SCLC), bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma,meningioma, melanoma, neuroblastoma, retinoblastoma, lung cancer,squamous cell carcinoma, adenocarinoma, large cell carcinoma, colorectalcancer, ovarian cancer, ovarian adenocarcinoma, prostate cancer,myelodysplastic syndromes (MDS) and multiple myeloma.

The term “neoplasm” or “neoplastic” also means a cell or tissueexhibiting abnormal growth, including hyperproliferation or uncontrolledcell growth, that may be benign or cancerous. The development from anormal cell to a cell exhibiting a neoplastic phenotype is a multi-stepprocess. Cells developing a neoplastic phenotype or designated as of acancerous cell type generally exhibit an alteration of the normal cellcycle and altered cell death response. Generally, the changes that acell undergoes in developing to a tumor cell may be monitored at thecellular or DNA level.

Therefore, the terms “pre-neoplasm” or “pre-neoplastic” phenotype areconstrued for the purposes of the present invention to refer to a cellor tissue which exhibits changes at the DNA or cellular level thatevidence the ultimate progression of the cell or tissue to a neoplasticor cancerous phenotype. Pre-neoplastic conditions do not show evidenceof microinvasion or other hallmarks of cancer behavior. As with thedevelopment to neoplasia, pre-neoplastic cells may exhibit progressionthrough multiple steps. Although a pre-neoplastic cell may progress to aneoplastic stage, they may remain stable for an extended period of timeand may even regress. The development of pre-neoplasia is oftenassociated with environmental factors. Examples of pre-neoplasticconditions in noninvasive bladder cancer include diffuse cellular atypiaof the urothelium.

The term “proliferative disorder” refers to disorders that areassociated with some degree of abnormal cell proliferation.

The term “precancerous” refers to cells or tissues havingcharacteristics relating to changes that may lead to malignancy orcancer. Examples include adenomatous growths in colon, ovary, breast,tissues, or conditions, for example, dysplastic nevus syndrome, aprecursor to malignant melanoma of the skin. Examples also include,abnormal neoplastic, in addition to dysplastic nevus syndromes,polyposis syndromes, prostatic dysplasia, and other such neoplasms,whether the precancerous lesions are clinically identifiable or not.

A “precancerous lesion(s)” may refer to an epithelial precancerouslesion, which is a skin lesion that has a propensity to develop into acancerous condition. Epithelial precancerous skin lesions also arisefrom other proliferative skin disorders such as hemangiomas, keloids,eczema and papilloma virus infections producing verruca vulbaris,verruca plantaris and verruca planar.

The symptoms of the epithelial precancerous lesions include skin-coloredor red-brown macule or papule with dry adherent scales. Actinickeratosis is the most common epithelial precancerous lesion among fairskinned individuals. It is usually present as lesions on the skin whichmay or may not be visually detectable. The size and shape of the lesionsvaries. It is a photosensitive disorder and may be aggravated byexposure to sunlight. Bowenoid actinic keratosis is another form of anepithelial precancerous lesion. In some cases, the lesions may developinto an invasive form of squamous cell carcinoma and may pose asignificant threat of metastasis. Other types of epithelial precancerouslesions include hypertrophic actinic keratosis, arsenical keratosis,hydrocarbon keratosis, thermal keratosis, radiation keratosis, viralkeratosis, Bowen's disease, erythroplaquia of queyrat, oralerythroplaquia, leukoplakia, and intraepidermal epithelialoma.

By “inhibits the growth of a neoplasm” is meant measurably slowing,stoping, or reversing the growth rate of the neoplasm or neoplasticcells in vitro or in vivo. Preferably, a slowing of the growth rate isby at least 20%, 30%, 40%, 50%, 60% or even 70% is achieved, over aperiod of treatment of six month as determined using a suitable assayfor determination of cell growth rates (e.g., a cell growth assaydescribed herein). Typically, a reversal of growth rate is accomplishedby initiating or accelerating necrotic or apoptotic mechanisms of celldeath in the neoplastic cells, resulting in shrinkage of the neoplasm.

The term “complete molecular response” means that molecular response asthe eIF4E gene product moving and going down (at least inleukemia-responses may be different in other cancers i.e. cancers withless nuclear eIF4E may not have pronounced movement to the cytoplasmetc.). Targeting eIF4E activity should also be there (looking at exportand translation targets); In leukemia as referred to herein, meanscomplete molecular response as eIF4E moving, eIF4E going down andtargeting the eIF4E gene product function.

By “an effective amount”, “a neoplasm treating amount”, “a pre-neoplasmtreating amount”, “a proliferative treating amount” or by “aprecancerous lesion treating amount” is meant the amount of a compoundor a combination of compounds required to treat or prevent a disease ina clinically relevant manner. An effective amount or a treating amountof a compound varies depending upon the disease being treated, themanner of administration, and the age, body weight, and general healthof the patient. Ultimately, the prescribers will decide the appropriateamount and dosage regimen according to good medical practice.

The term “therapeutically effective amount” intends to describeconcentrations or amounts of compounds according to the presentinvention which are therapeutically effective in treating neoplasm (I.e.tumors, cancers, etc.), pre-neoplasm, proliferative disorders, and/orprecancerous lesions or the various conditions or disease statesincluding hyperproliferative cell growth.

The term “effective amount” shall mean an amount or concentration of acompound or composition according to the present invention which iseffective within the context of its administration, which may beinhibitory, prophylactic and/or therapeutic. Compounds according to thepresent invention are particularly useful for providing favorable changein the disease or condition treated, whether that change is a remission,a decrease in growth or size of cancer or a tumor or other effect of thecondition or disease to be treated, a favorable physiological result ora reduction in symptomology associated with the disease or conditiontreated.

The term “administering” refers to a method of giving a composition ofthe invention to a patient, by a route such as inhalation, ocularadministration, nasal instillation, parenteral administration, dermaladministration, transdermal administration, buccal administration,rectal administration, sublingual administration, perilingualadministration, nasal administration, topical administration and oraladministration. Parenteral administration includes intrathecal,intraarticular, intratumoral, intravenous, intraperitoneal,subcutaneous, and intramuscular administration. The optimal method ofadministration of a drug or drug combination to treat a particulardisease can vary depending on various factors, e.g., the oralbioavailability of the drug(s), the anatomical location of the diseasetissue, and the severity of disease.

The hedgehog (Hh) signaling pathway plays an important role inembryogenesis across multiple species. Its activity is reduced or absentin adult organisms. However, activation of the pathway has been shown tobe a factor in the development of a number of human malignancies andinhibition of the pathway is being investigated as a potential treatmentfor multiple cancers.

The term ‘western blot” refers to the analysis of protein(s) (orpolypeptides) immobilized onto a support such as nitrocellulose or amembrane. The proteins are run on polyacrylamide gels to separate theproteins, followed by transfer of the protein from the gel to a solidsupport, such as nitrocellulose, polyvinylidene fluoride (PVDF) or asimilar membrane. The immobilized proteins are then exposed toantibodies with reactivity against an antigen of interest. The bindingof the antibodies can be detected by various methods, including the useof radio-labeled antibodies or chemiluminescence.

Other features and advantages of the invention will be apparent from thefollowing detailed description, the drawings, and the claims.

Pharmacological Inhibition of eIF4E by Ribavirin

Ribavirin has been found to act as a competitive inhibitor of the m⁷Gcap and thereby inhibiting eIF4E functions in both the export andtranslation of sensitive transcripts. NMR, fluorescence, and massspectrometry studies indicate that ribavirin directly binds eIF4E. ³Hribavirin binds to eIF4E in living cells as shown by the 14-foldenrichment of ³H ribavirin in eIF4E immunoprecipitations relative tocontrols. The active metabolite of ribavirin, ribavirin triphosphate(RTP), binds eIF4E with a similar affinity as m⁷GTP and impedes mRNAexport of eIF4E targets and translation of vascular endothelial growthfactor (VEGF) and ornithine decarboxylase (ODC) but not the export ortranslation of eIF4E insensitive transcripts such as glyceraldehyde3-phosphate dehydrogenase (GAPDH). Ribavirin sensitivity parallels eIF4Esensitivity and inhibits both the nuclear and cytoplasmic functions ofeIF4E. Importantly, ribavirin affects primary AML specimens withelevated eIF4E more than specimens with normal eIF4E levels. In total,it appears that these AML specimens have developed an “oncogeneaddiction” to eIF4E making them more sensitive to inhibition of eIF4Ethan specimens with normal eIF4E levels. The clinical utility ofribavirin treatment in M4/M5 AML patients was investigated.

The effectiveness of chemotherapy is limited in some cases because theleukemia cells become resistant to it or have a resistance due to priortherapies or a de novo resistance (primary). As discussed above, theApplicant tested the efficacy of ribavirin treatment in patients in aCanada-wide clinical trial, and observed striking improvement with thepatients, however, all eventually developed resistance to ribavirin(Assouline et al., Blood, 2009).

Usually, chemotherapy is given in cycles, with each period of treatmentfollowed by a rest period to allow the body time to recover. Therefore,researchers are looking at ways to prevent or reverse this resistance byusing other drugs along with chemotherapy.

Cooperation Between the Inhibitor of eIF4E and the Hedgehog PathwayInhibitor

The eukaryotic translation initiation factor eIF4E gene is elevated inM4 and M5 acute myeloid leukemia (AML). The oncogenic potential of eIF4Edepends on its ability to bind the m⁷G cap on the 5′ end of mRNAs.Applicant discovered that ribavirin acts as a competitive inhibitor ofthe cap thereby inhibiting eIF4E's activities. The phase II ribavirinmonotherapy trial in poor prognosis AML patients demonstrated thatribavirin treatment targeted the eIF4E gene product activity and thiscorrelated with clinical benefit including remissions. To improveclinical outcomes, the features that lead to primary and acquiredribavirin resistance were examined as were heterogeneity in patientresponse, assessing new contexts for ribavirin use and determining theefficacy of combining ribavirin with a hedgehog pathway inhibitor and/orwith low dose cytarabine (Ara-C®) in patients.

To overcome the resistance problem due to prior anti-neoplastictherapies or a de novo resistance in the treatment with ribavirin, andaccording to the present invention, there is provided a combinationtherapy for treating patients having an affliction selected from thegroup consisting of: a neoplasm, a pre-neoplasm, a proliferativedisorder, and a precancerous lesion.

To improve clinical outcomes by acquired ribavirin resistance,heterogeneity in patient's response and resistance in patient specimenswas explored. In a preferred embodiment, the inventors assessed newcontexts for ribavirin use, including the efficacy of combining aninhibitor of the eIF4E gene product with a hedgehog pathway inhibitorand/or a chemotherapeutic agent in patients.

Two distinct mechanisms were identified underlying resistance. First,ribavirin uptake is impaired through loss of its nucleoside transporterand/or adenosine kinase. Second, elevation of the sonic hedgehogtranscription factor Gli-1 leads to activation of UGT1A enzymes andsubsequent glucurondiation of ribavirin, thereby preventing itsassociation with eIF4E. Gli-1 overexpression is sufficient to impartresistance. Genetic knockdown of Gli-1, or pharmacological inhibition ofGli-1 with GDC-0449, reverts resistance, correlating with reappearanceof ribavirin-eIF4E complexes. In patient specimens, clinical resistancecorrelated with elevated Gli-1 levels. Thus, resistance to a targetedtherapy can be driven by chemical modification of the drug rather thansolely by mutation of the target protein. Gli-1 also has proliferativecapacity and could drive oncogenesis independently of eIF4E in resistantcells due to genetic re-wiring.

The combination therapy according to the present invention comprising aninhibitor of eIF4E, a hedgehog pathway inhibitor and/or achemotherapeutic agent overcomes the resistance developed in patientsduring anti-neoplastic treatment.

The effects of the combined treatment of an inhibitor of eIF4E,preferably ribavirin with a hedgehog pathway inhibitor, preferablyGDC-0449, were examined. GDC-0449 is a small molecule inhibitor ofHedgehog-Gli pathway being developed for potential use in cancertreatment.

As discussed above, Gli-1 lead to the loss of ribavirin-eIF4Einteraction and thus resistance in F10R cells, that the UGT1A family ofproteins was highly elevated. The Applicant has examined that there is alink between Gli-1 and UGT and that this leads to glucurondiation ofribavirin, losing the interaction. The applicant has examinedpretreating the F10R cells with GDC-0449 and then adding the ribavirin.

The applicant has examined whether the inhibitor of eIF4E, preferablyribavirin and a hedgehog pathway inhibitor, preferably GDC-0449,cooperated in poor prognosis AML patient specimens.

According to the present invention, novel combinatory drug therapy isuseful due to the cooperation between the inhibitor of eIF4E, thehedgehog pathway inhibitor, and, if necessary, the chemotherapeuticagent, which destroys cancer cells by interfering with DNA synthesis.

Cooperation Between the Inhibitor of eIF4E and the ChemotherapeuticAgent

Several models of collaboration between these drugs was determined,namely:

-   -   Ribavirin inhibits eIF4E functions and cytarabine (Ara-C®)        interferes with nucleic acid synthesis or nucleotide synthesis,        being an inhibitor of DNA polymerase and blocking DNA synthesis,        but having no effect on RNA or protein synthesis;    -   Cytarabine incorporated into RNA and DNA interfering with chain        elongation; ribavirin acts as a competitive inhibitor of the m⁷G        cap;    -   Cytarabine (Ara-C®) is cytotoxic and is highly specific for the        S phase of the cell cycle, whereas ribavirin is cytostatic;    -   Cytarabine (Ara-C®) can increase ribavirin's inhibition of its        targets by either blocking proper 5′cap formation on these mRNAs        and/or by incorporating into cellular RNAs and further        inhibiting their translation or export;    -   Cytarabine (Ara-C®) exhibits cell phase specificity, primarily        killing cells undergoing DNA synthesis (S-phase) and under        certain conditions blocking the progression of cells from the G1        phase to the S-phase (The Extra Pharmacopoeia, 30th ed); and    -   Ribavirin induces a G1/S arrest in many cell types (Kentsis et        al., 2004). Thus, ribavirin can collaborate in this way with        cytarabine (Ara-C®).

The effects of the combined treatment of an inhibitor of eIF4E,preferably ribavirin with an antimetabolite, preferably cytarabine(Ara-C®), have been examined. Cytarabine (Ara-C®) is a pyrimidinenucleoside analog that inhibits the synthesis of DNA, and has shownspecificity for the S phase of the cell cycle. It is metabolizedintracellularly into its active triphosphate form (cytosine arabinosidetriphosphate). Further this metabolite damages DNA by multiplemechanisms, including the inhibition of alpha-DNA polymerase, inhibitionof DNA repair through an effect on beta-DNA polymerase, andincorporation into DNA.

The Applicant has examined whether the inhibitor of eIF4E, preferablyribavirin and a chemotherapeutic agent, preferably cytarabine (Ara-C®)cooperated in poor prognosis AML.

It is not uncommon in cancer treatments to have mixtures of three ormore drugs. In this connection, other agents may be used in conjunctionwith the combination of inhibitor of eIF4E and the hedgehog pathwayinhibitor. For example, the composition according to the presentinvention may further comprise at least another pharmaceutically activesubstance. Other pharmaceutically active substance(s), include, and arenot limited to: topoisomerase inhibitors, NFKB inhibitors, hedgehogpathway inhibitors, methyltransferase inhibitors, etc.

Preferably, the inhibitor of eIF4E is ribavirin, the hedgehog pathwayinhibitor is GDC-0449 and the chemotherapeutic agent is selected fromthe category of antimetabolites, more preferably, from the class ofpyrimidine antagonists, and most preferably, is cytarabine (Ara-C(Registered trademark)).

Indications for Treatment

The compositions of the present invention are preferably aimed attreating conditions which involve undesirable or uncontrolled cellproliferation. Such conditions include neoplasms, pre-neoplasms,proliferative disorders and precancerous lesions. In a preferredembodiment, the neoplasm is cancer.

Preferably, the cancer is selected from the group consisting ofleukemia, acute myeloid leukemia, acute myelocytic leukemia, acutemyeloblastic leukemia, acute promyelocytic leukemia, acutemyelomonocytic leukemia, acute monocytic leukemia, acuteerythroleukemia, chronic leukemia, chronic myelocytic leukemia, chroniclymphocytic leukemia, polycythemia vera, lymphoma, Hodgkin's disease,non-Hodgkin's disease lymphoma, Waldenstrom's macroglobulinemia, heavychain disease, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lyinphangiosarcoma, lymphangioendotheliosarcoma, synovioma,mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, coloncarcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostatecancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma,sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor,cervical cancer, uterine cancer, testicular cancer, lung carcinoma,small cell lung carcinoma, bladder carcinoma, epithelial carcinoma,glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma,pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma,schwannoma, meningioma, melanoma, neuroblastoma, retinoblastoma, lungcancer, squamous cell carcinoma, adenocarcinoma, large cell carcinoma,colorectal cancer, ovarian cancer, ovarian adenocarcinoma, prostatecancer, myelodysplastic syndromes, and multiple myeloma.

According to the present invention, the acute myeloid leukemia is acutemyeloid leukemia M4 or acute myeloid leukemia M5 or another AML subtypecharacterized by atypical elevation of the e1F4E gene product.

Other types of cancers which could be potentially treated include butare not limited to leukemia, acute myeloid leukemia, acute myelocyticleukemia, acute myeloblastic leukemia, acute promyelocytic leukemia,acute myelomonocytic leukemia, acute monocytic leukemia, acuteerythroleukemia, chronic leukemia, chronic myelocytic leukemia, chroniclymphocytic leukemia, polycythemia vera, lymphoma, Hodgkin's disease,non-Hodgkin's disease lymphoma, Waldenstrom's macroglobulinemia, heavychain disease, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lyinphangiosarcoma, lymphangioendotheliosarcoma, synovioma,mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, coloncarcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostatecancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma,sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor,cervical cancer, uterine cancer, testicular cancer, lung carcinoma,small cell lung carcinoma, bladder carcinoma, epithelial carcinoma,glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma,pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma,schwannoma, meningioma, melanoma, neuroblastoma, retinoblastoma, lungcancer, squamous cell carcinoma, adenocarinoma, large cell carcinoma,colorectal cancer, ovarian cancer, ovarian adenocarcinoma, prostatecancer, myelodysplastic syndromes and multiple myeloma.

Generally, cells in a benign tumor retain their differentiated featuresand do not divide in a completely uncontrolled manner. A benign tumor isusually localized and non metastatic. Specific types of benign tumorsthat can be treated using the present invention include hemangiomas,hepatocellular adenoma, cavernous haemangioma, focal nodularhyperplasia, acoustic neuromas, neurofibroma, bile duct adenoma, bileduct cystanoma, fibroma, lipomas, leiomyomas, mesotheliomas, teratomas,myxomas, nodular regenerative hyperplasia, trachomas and pyogenicgranulomas.

In a malignant tumor, cells become undifferentiated, do not respond tothe body's growth control signals, and multiply in an uncontrolledmanner. The malignant tumor is invasive and capable of spreading todistant sites (metastasizing). Malignant tumors are generally dividedinto two categories: primary and secondary. Primary tumors arisedirectly from the tissue in which they are found. A secondary tumor, ormetastasis, or even infiltrating leukemia cells, is a tumor which isoriginated elsewhere in the body but has now spread to a distant organ.The common routes for metastasis are direct growth into adjacentstructures, spread through the vascular or lymphatic systems, andtracking along tissue planes and body spaces (peritoneal fluid,cerebrospinal fluid, etc.). Malignant leukemia cells can infiltrate intoother tissues.

Specific types of cancers or malignant tumors, either primary orsecondary, that can potentially be treated using the present inventioninclude leukemia, breast cancer, skin cancer, bone cancer, prostatecancer, liver cancer, lung cancer, brain cancer, cancer of the larynx,gall bladder, pancreas, rectum, parathyroid, thyroid, adrenal, neuraltissue, head and neck, colon, stomach, bronchi, kidneys, basal cellcarcinoma, squamous cell carcinoma of both ulcerating and papillarytype, metastatic skin carcinoma, osteo sarcoma, Ewing's sarcoma,veticulum cell sarcoma, myeloma, giant cell tumor, small-cell lungtumor, gallstones, islet cell tumor, primary brain tumor, acute andchronic lymphocytic and granulocytic tumors, hairy-cell tumor, adenoma,hyperplasia, medullary carcinoma, pheochromocytoma, mucosal neuronms,intestinal ganglloneuromas, hyperplastic comeal nerve tumor, marfanoidhabitus tumor, Wilm's tumor, seminoma, ovarian tumor, leiomyomatertumor, cervical dysplasia and in situ carcinoma, neuroblastoma,retinoblastoma, soft tissue sarcoma, malignant carcinoid, topical skinlesion, mycosis fungoide, rhabdomyosarcoma, Kaposi's sarcoma, osteogenicand other sarcoma, malignant hypercalcemia, renal cell tumor,polycythermia vera, adenocarcinoma, glioblastoma multiforma, leukemias,lymphomas, malignant melanomas, epidermoid carcinomas, and othercarcinomas and sarcomas.

Hematologic disorders include abnormal growth of blood cells, which canlead to dysplastic changes in blood cells and hematologic malignanciessuch as various leukemias. Examples of hematologic disorders include butare not limited to acute myeloid leukemia, acute promyelocytic leukemia,acute lymphoblastic leukemia, chronic myelogenous leukemia, themyelodysplastic syndromes (MDS), multiple myeloma and sickle cellanemia.

Acute myeloid leukemia (AML) is the most common type of acute leukemiathat occurs in adults. Several inherited genetic disorders andimmunodeficiency states are associated with an increased risk of AML.These include disorders with defects in DNA stability, leading to randomchormosomal breakage, such as Bloom's syndrome, Fanconi's anemia,Li-Fraumeni kindreds, ataxia-telangiectasia, and X-linkedagammaglobulinemia.

Acute promyelocytic leukemia (APML) represents a distinct subgroup ofAML. This subtype is characterized by promyelocytic blasts containingthe 15; 17 chromosomal translocation. This translocation leads to thegeneration of the fusion transcript comprised of the retinoic acidreceptor and the PML gene product.

Acute lymphoblastic leukemia (ALL) is a heterogenerous disease withdistinct clinical features displayed by various subtypes. Reoccurringcytogenetic abnormalities have been demonstrated in ALL. The most commoncytogenetic abnormality is the 9; 22 translocation. The resultantPhiladelphia chromosome represents poor prognosis of the patient.Chronic myelogenous leukemia (CML) is a clonal myeloproliferativedisorder of a pluripotent stem cell. CML is characterized by a specificchromosomal abnormality involving the translocation of chromosomes 9 and22, creating the Philadelphia chromosome. Ionizing radiation isassociated with the development of CML. Chronic phase CML is oftensuccessfully treated with Gleevec®. However, when this converts to blastcrisis CML, Gleevec® is not effective. Our previous studies indicatethat eIF4E levels are elevated in blast crisis CML, but not in thechronic phase. This means that blast crisis CML patients could becandidates for the combination therapy according to the presentinvention.

The myelodysplastic syndromes (MDS) are heterogeneous clonalhematopoietic stem cell disorders grouped together because of thepresence of dysplastic changes in one or more of the hematopoieticlineages including dysplastic changes in the myeloid, erythroid, andmegakaryocytic series. These changes result in cytopenias in one or moreof the three lineages. Patients afflicted with MDS typically developcomplications related to anemia, neutropenia (infections), orthrombocytopenia (bleeding). Generally, from about 10% to about 70% ofpatients with MDS develop acute leukemia. MDS patients could becandidates for this therapy.

Routes of Administration and Dosing Regimen

A number of routes of administration and formulations may be used in thecombination therapies of the present invention. The combination oftherapeutic treatment according to the present invention may beadministered in combination with one or more conventional pharmaceuticalagents.

These additional agents may include additional compounds according tothe invention, or one or more other pharmaceutically active agents.

The compound may be administered or coadministered orally, parenterally,intraperitoneally, intravenously, intraarterially, transdermally,sublingually, intramuscularly, rectally, transbuccally, intranasally,liposomally, via inhalation, vaginally, intraoccularly, via localdelivery (for example by catheter or stent), subcutaneously,intraadiposally, intraarticularly, or intrathecally. More particularly,other forms of administration include, for example: inhalation, ocularadministration, nasal instillation, parenteral administration, dermaladministration, transdermal administration, buccal administration,rectal administration, sublingual administration, perilingualadministration, nasal administration, topical administration, and oraladministration.

The compounds according to the present invention may also beadministered or co-administered, sequentially or simultaneously, inimmediate release, delayed release or even slow release dosage forms.

The inhibitor of eIF4E, the hedgehog pathway inhibitor, and thechemotherapeutic agent, according to the present invention, areadministered sequentially or simultaneously. The compound according tothe present invention may be administered by a variety of routes, andmay be administered or co-administered in any conventional dosage form.

The composition, according the present invention, is in a unit dosageform. Co-administration in the context of this invention is defined tomean the administration of more than one therapeutic in the course of acoordinated treatment to achieve an improved clinical outcome. Suchco-administration may also be coextensive, that is, occurring duringoverlapping periods of time. For example, the chemotherapeutic agent maybe administered to a patient before, concomitantly, or after theinhibitor of eIF4E is administered. In a preferred embodiment, thepatient may be pretreated with the hedgehog pathway inhibitor and thentreated with the inhibitor of eIF4E (e.g., ribavirin).

The amounts of the therapeutic agents present in the compositions canvary, according to determinations made by a person skilled in the art,but preferably are in amounts effective to create a cytotoxic orcytostatic effect at the desired site. Preferably, these total amountsare less than the total amount adding the maximum tolerated dose foreach of the Ara-C®, the eIF4E inhibitor, and the hedgehog pathwayinhibitor, and more preferably less than the total amount added forindividual administration of each of these inhibitors. In a preferredembodiment, the amount of therapeutic agent(s), i.e., an inhibitor ofeIF4E, hedgehog pathway inhibitor and/or a Ara-C® are deemed to be in aneffective amount for treating the indication, for example: a neoplasm(i.e. cancer or, more particularly, acute myeloid leukemia), apre-neoplasm, a proliferative disorder, or a precancerous lesion.Preferably, for the dosage form, appropriate release times can vary, butpreferably should last from about 1 hour to about 6 months and mostpreferably from about 1 week to about 4 weeks. Formulations includingthe compositions according to the present invention can vary, asdeterminable by a person skilled in the art, according to the particularsituation, and as generally taught herein.

In a preferred embodiment, the inhibitor of eIF4E (preferably ribavirin)is administered in an amount between about 500 mg to 4400 mg per day,and more preferably, in the range between about 1000 mg to about 4400 mgper day. The hedgehog pathway inhibitor, such as GDC-0449, is preferablyadministered at 3 nM for 2 days prior to the start of ribavirintreatment. The Ara-C® is preferably administered in an amount up toabout 100 mg/m². This administration can preferably last for up to 7days every 4 weeks, on a repetitious basis if required. Preferably, theAra-C® is administered in an amount sufficient to repress tumorigenicityof cells. More preferably, the Ara-C® is administered in a low dose. Inthis connection, a low dose of Ara-C® preferably ranges from about 5mg/day to about 20 mg twice a day.

Also according to an embodiment of the present invention, after thetreatment with GDC-0449 and ribavirin, the patient may be furthertreated with Ara-C® and various anticancer agents described above.

According to the present invention, there is provided a method toadminister to a patient a therapeutically effective amount of aninhibitor of eIF4E, a therapeutically effective amount of a hedgehogpathway inhibitor and a therapeutically effective amount of achemotherapeutic agent.

Preferably, the hedgehog pathway inhibitor is GDC-0449, using 3 nM whichwas reported to be the plasma levels according to Roche.

More preferably, a therapeutically effective amount of the inhibitor ofeIF4E and the therapeutically effective amount of GDC-0449 areadministrated simultaneously or sequentially in resistant cell lines.

According to the present invention, there is provided a method toadminister to a patient a therapeutically effective amount of theinhibitor of eIF4E and the therapeutically effective amount of thehedgehog pathway inhibitor prior to initiating administration of thechemotherapeutic agent. Preferably, the inhibitor of eIF4E and saidhedgehog pathway inhibitor are administrated concurrently with thechemotherapeutic agent. Also preferably, the inhibitor of eIF4E and saidhedgehog pathway inhibitor are administrated sequentially with thechemotherapeutic agent.

According to the present invention, there is provided a method toadminister to treat a patient with a combination therapy for treatingpatients having a neoplasm or a proliferative disorder, comprisingadministering to a patient a therapeutically effective amount of thehedgehog pathway inhibitor in the presence of a therapeuticallyeffective amount of ribavirin wherein said combination therapy revertsresistance developed in patients during anti-neoplastic treatment or inpatients with primary resistance.

Owing to the sensitizing effects of the combination therapy on the cellsto cell death or senescence or some permanent cell cycle arrested state,the dosage of anticancer agents used for the treatment may be lower thanthat used in a conventional cancer treatment regimen. Thus, a betterclinical outcome may be achieved by using the compositions and methodsof the present invention.

The present invention provides for:

-   -   the use of combination therapy for treating patients having a        neoplasm or a proliferative disorder, comprising an inhibitor of        the eIF4E gene product, a hedgehog pathway inhibitor, and a        chemotherapeutic agent, wherein said combination therapy        overcome resistance developed in patients during anti-neoplastic        treatment;    -   the use of combination therapy in treating the neoplasm or        proliferative disorder comprising administering to a patient a        therapeutically effective amount of an inhibitor of the eIF4E        gene product, a therapeutically effective amount of a hedgehog        pathway inhibitor with or without a therapeutically effective        amount of a chemotherapeutic agent;    -   a method of use for a combination therapy in treating patients        having a neoplasm or a proliferative disorder, comprising an        inhibitor of the eIF4E gene product and the chemotherapeutic        agent, wherein said combination therapy overcomes resistance        developed in patients during anti-neoplastic treatment;    -   a method of use a combination therapy for treating a neoplasm or        a proliferative disorder, wherein said method comprises        administering a therapeutically effective amount of an inhibitor        of the eIF4E gene product, a therapeutically effective amount of        a hedgehog pathway inhibitor and a therapeutically effective        amount of a chemotherapeutic agent, wherein said combination        therapy overcomes resistance developed in patients during        anti-neoplastic treatment from previous therapies, from        ribavirin therapy or de novo;    -   the use of combination therapy for treating patients having a        neoplasm or a proliferative disorder, comprising an inhibitor of        the eIF4E gene product, a hedgehog pathway inhibitor and a        chemotherapeutic agent, wherein said combination therapy        minimizes or prevents the growth of resistant cells developed in        patients during anti-neoplastic treatment; inhibition of the        hedgehog pathway to revert resistance and/or decrease        proliferation; and    -   a method of use of a combination therapy for treating a        pre-neoplasm or a precancerous lesion, wherein said method        comprising administering a therapeutically effective amount of        an inhibitor of the eIF4E gene product, a therapeutically        effective amount of a hedgehog pathway inhibitor and a        therapeutically effective amount of a chemotherapeutic agent,        wherein said combination therapy overcome resistance developed        in patients during anti-neoplastic treatment from previous        therapies, from ribavirin therapy or de novo.

In a preferred embodiment, the preneoplasm or precancerous lesion isselected from the group consisting of: proliferative disorders that leadto the development of solid or hematological neoplasms and preneoplasmsor precancerous lesions that may evolve into a neoplasm.

The following examples are intended to illustrate, but not limit theinvention.

EXAMPLES

According to the present invention, there is provided a use of acombination therapy for treating patients having a neoplasm,proliferative disorder, pre-neoplasm or precancerous lesion comprisingan inhibitor of eIF4E, a hedgehog pathway inhibitor, and achemotherapeutic agent, wherein said combination therapy overcomesresistance developed in patients during anti-neoplastic treatment fromprevious therapies, from ribavirin therapy or de novo.

The treatment comprises administering a therapeutically effective amountof ribavirin, as an inhibitor of the eIF4E gene product; administering atherapeutically effective amount of a hedgehog pathway inhibitor, whichis GDC-0449; and administering a therapeutically effective amount ofchemotherapeutic agent, which is cytarabine (Ara-C (Registeredtrademark)).

The patients can be administered at least between 500 mg/day to 4400mg/day of ribavirin and at least between 50 mg/m2 to 150 mg/m2 ofcytarabine. More preferably, a patient can be administered at leastbetween 1000 and 2800 mg/day of ribavirin and up to 100 mg/m2 ofcytarabine. In even a more preferred embodiment, the inhibitor of eIF4Eis administered in an amount between 1000 to 4400 mg per day and thecytarabine (Ara-C (Registered trademark)) is administered in an amountfrom 10 mg/day to 20 mg twice a day.

The effects of either drug treatment alone or in combination, for AMLspecimens, is disclosed hereinabove as well as in FIG. 1. The eIF4E geneproduct is substantially up-regulated and forms abnormally large nuclearbodies in a subset of AML (FAB subtype M4/M5) and blast crisis CMLprimary specimens (FIG. 1).

Further, eIF4E dependent mRNA export of targets such as cyclin D1 issubstantially up-regulated leading to increased protein levels for thesetargets and subsequent proliferation.

Phase II Ribavirin Monotherapy Trial in Poor Prognosis AML Patients

Ribavirin treatment targeted the eIF4E gene product activity in patientswith relapsed/refractory M4/M5 AML or who were deemed unable to undergostandard chemotherapy.

In terms of clinical response, 1 CR (complete remission), 2 PR (partialremission), 3 BR (blast response), 6 SD (stable disease), 3PD(progressive disease) were observed out of 15 evaluable patients. Nodrug related toxicity was observed in any of the patients. The molecularanalyses indicated that ribavirin treatment targeted the eIF4E geneproduct activity and this correlated with response. For instance, themRNA export activity of the eIF4E gene product was directly monitoredand it was shown that, after 28 days of oral ribavirin, this wassubstantially reduced. A biphasic molecular response was observed, whereinitially the eIF4E gene product was dramatically re-distributed to thecytoplasm in patients that responded. This was followed by a drop ineIF4E RNA and protein levels (up to one 28 day cycle). This conclusivelysupports that ribavirin directly targets the eIF4E gene product activityin patients and this correlated with clinical benefit. However, allresponding patients eventually became resistant to ribavirin.

Phase I Ribavirin and Low Dose ARA-C® Trial in Poor Prognosis AML

Phase I trial was carried out to establish the safety of a combinationof oral ribavirin and subcutaneous low dose Ara-C® in the same patientpopulation. These compounds cooperated in colony growth assays ofprimary specimens. Ara-C® was kept constant (except in case of toxicity)and ribavirin doses elevated in a 3+3 trial design for the phase I arm.In the first dose level, it was noted that ribavirin plasma levels weresubstantially lower than was observed in the monotherapy trial. As knownfrom prior art, other oral compounds also had their absorption reducedby subcutaneous Ara-C® including digoxin.

In the monotherapy trial at 2000 mg/day of ribavirin, plasma levels ofaround 20-30 μM ribavirin (determined by mass spectrometry) wereachieved, which corresponded to a therapeutic range whereas in thecombination trial ribavirin plasma levels ranged from only 4-10 μM. Inthe monotherapy trial at 2800 mg/day, plasma levels of 20 μM ribavirinwere achieved.

In phase I combination trial, 5 patients achieved plasma levels of 20 μMribavirin or higher. Of these, has been observed a complete molecularresponse for the CR, BR, SD and none for the 2 PD indicating thatribavirin levels were sufficient to effect the eIF4E gene productlocalization and levels. The CR had secondary AML due to breast cancertherapy and had some toxicities due to Ara-C®, and thus the Ara-C®levels were lowered to 10 mg/day. This correlated with a near doublingof plasma ribavirin levels and achievement of CR. After 6 cycles, Ara-C®was discontinued and patient remains in CR on ribavirin alone for thepast twelve cycles (totaling 18 cycles).

Heterogeneity in Patient Response

It was observed that patients from both trials that had a completemolecular response to ribavirin (lowering of the eIF4E levels after aninitial relocalization of the eIF4E gene product) achieved CR, PR, BR orSD (but NOT PD). This heterogeneity suggests that eIF4E is more centralin driving the leukemia of the best responding patients than others.This is consistent with the theory that the entire leukemia cellpopulation has not coupled its survival to the same signalling pathways.Patients with a greater number of eIF4E dependent leukemia cells wouldbe expected to respond better. There is the distinct possibility thatother pathways are needed to cooperate with the eIF4E gene product totransform cells and the nature of these pathways may be heterogeneousand differentially affect ribavirin response. The sonic hedgehog pathwayis one such pathway.

It was also observed that there was no correlation between Flt3 or NPMstatus and response. Alternatively or in addition, some patients (whoare mostly heavily pre-treated) may be on the verge of resistance whenthey enter the trial and that ribavirin treatment leads to a finalselection of a resistant population in terms of the ENT1 nucleosidetransporter, used by both ribavirin and Ara-C®.

Ribavirin Heterogeneity

To understand the heterogeneity in clinical response, it is critical tostudy patient specimens to monitor changes in gene expression as afunction of treatment, response, and resistance.

Heterogeneity in response and resistance in patient specimens wasexplored. The gene expression profile of a variety of patient specimenswas examined by deep sequencing to assess differences before treatment,during response, and at relapse as well as comparing responders tonon-responders.

Factors that contribute to resistance in resistant cell lines wereexamined and candidates from these studies were monitored in patientspecimens. Such markers should help predict which patients aremost/least likely to benefit from ribavirin treatment. Deep sequencinganalysis revealed that Gli-1, the sonic hedgehog transcription factor,was highly elevated in cell lines that had normal uptake of ribavirin,but had no ribavirin response in terms of growth or the eIF4E geneproduct activity. Indeed in these cells, the ribavirin-eIF4E interactionwas lost despite the fact that the eIF4E gene product was not mutated.

Ribavirin Resistance Correlates with Loss of EIF4E Targeting

It was assessed whether ribavirin is still targeting eIF4E activity inresistant cells (FIG. 4). Protein levels of well-established eIF4E mRNAexport and translation targets, including cyclin D1 and VEGF weremonitored as a function of ribavirin treatment. In parental cell lines,ribavirin treatment reduces levels of these proteins whereas inresistant cell lines, ribavirin treatment no longer affects these. Therewas no change in the total levels of eIF4E between resistant andparental cell lines, as observed in other cell line systems.

It was determined whether ¹⁴C-ribavirin could immunoprecipitate witheIF4E in the F10R and F100R resistant cell lines. It is noted that the¹⁴C or ³H label is at position 5 of the triazole ring i.e. the activemoiety of ribavirin. In parental cell lines, ¹⁴C ribavirin is enrichedin the eIF4E immunoprecipitated fraction by about 20 fold relative tothe IgG control, whereas ¹⁴C ribavirin does not immunoprecipitate witheIF4E in the F10R or F100R cell lines (FIG. 4). Sequencing analysisindicated that there were no mutations in the coding region of eIF4E inF10R and F100R cells.

Defects in Ribavirin Uptake and Metabolism

The uptake of ³H-ribavirin as a potential mechanism for ribavirinresistance was examined (FIG. 5). Previous studies indicate that maximaluptake is around 8-10 hours after ribavirin treatment. Uptake from 0 to16 hours at different ribavirin concentrations was monitored. ParentalFaDu and THP1 cells showed uptake profiles similar to those reportedpreviously. The F100R, T10R and T20R cell lines all showed severedefects in ribavirin uptake. Interestingly, F10R cells, althoughresistance to ribavirin had uptake that was indistinguishable fromparental FaDu cells. Thus, there are, at least, two major forms ofribavirin resistance, type I, which has a defect in net uptake, and typeII which is characterized by normal uptake but loss of the interactionof ribavirin with eIF4E.

Ribavirin is taken up by all cells via the equilibrative nucleosidetransporter ENT1 and once in the cell is phosphorylated to ribavirinmonophosphate using adenosine kinase (ADK). Subsequent phosphorylationto its active metabolite, ribavirin triphosphate (RTP) occurs via a widevariety of cellular kinases. Real time quantitative PCR (RT-qPCR)methods and western blot analyses revealed that ADK and ENT1 werealtered in the resistant THP cells but only ADK was altered in F100Rcells. In F100R cells, ADK RNA levels are reduced 30 fold as assessed byRT-qPCR while ENT1 RNA levels are not changed. At the protein level, ADKprotein levels are substantially reduced relative to parental cellsindicating that ADK is reduced at the transcript level while ENT1 isdownregulated post-transcriptionally. In THP1 cells, where ADK wasdownregulated at the transcript level whereas ENT1 is reducedpost-transcriptionally in THP1 resistant cells, with the exception ofT2×20R cells. T2×20R and F100R cells have normal protein and RNA levelsof ENT1 (compared to parental cell lines) but do have very low levels ofADK RNA and protein. ADK defects alone are sufficient to reduce netuptake of ribavirin because the unphosphorylated ribavirin can bereadily exported.

The Sonic Hedgehog Transcription Factor GLI1 Underlies Type IIResistance

F10R cells had no defect in ribavirin uptake and had normal levels ofADK and ENT1 RNA and proteins. Ribavirin did not associate with eIF4E inthese cells, or target eIF4E activity. (FIG. 6) To determine themolecular underpinnings for this form of resistance, deep sequencing wasused to compare F10R transcripts levels with those of F100R cells andwith low and high passage parental FaDu cells.

Cross comparison of F10R versus all other groups indicated that lessthan 30 transcripts were different using a padj cutoff value of 0.01.(Table 1). The statistically most significant difference was gliomaassociated protein, Gli-1, which was elevated 20 fold in F10R cellsversus the others. It was shown that Gli-1 was elevated at the RNA andprotein levels in F10R cells using real time PCR and western analysis(FIG. 7). Elevation of Gli-1, the downstream transcription factor forsignalling via the sonic hedgehog pathway, is associated with drivingproliferation and resistance in other systems. The mechanism for thelatter is usually attributed to the proliferative capacity of thissignaling pathway.

TABLE 1 Gli-1 mRNA levels relative to multiple healthy volunteersPatient Relapse/ # Response Before During response EOT 17 BR 20 5.8 2610 BR 1.3 1.6 3.6 6 BR 2.0 n/a 15 8 PR n/a 0.1 2.0 11 CR n/a 0.5 1.5 18SD 6.4 2.7*20% drop in blasts n/a 13 SD 1.4 2.6* blasts elevated butstill SD 5.3 3 SD 7 12* blasts remained same 23 9 PD 800 — n/a 19 PD 19— 36Preliminary Results from Monotherapy Trial for Patients Finishing OneCycle.

It was examined whether Gli-1 overexpression alone was sufficient toimpart resistance to ribavirin in FaDu cells. Gli-1 overexpressing cellswere virtually unaffected by the addition of 20 μM ribavirin (95% of theuntreated Gli-1 overexpressing cells) unlike the parental cells wheretreatment with 20 μM ribavirin reduced cell number by 50%. (FIG. 6).Gli-1 knockdown experiments were performed in parallel. Upon treatmentwith 20 μM ribavirin, growth of siRNA luciferase treated F10R cells wasunaffected. In contrast, RNAi mediated knockdown of Gli-1 re-sensitizesF10R cells to ribavirin with cell numbers being about 50% of these samecells without ribavirin. siRNA medicated knockdown of Gli-1 alone had noeffect on cell number for untreated F10R cells relative to controlsiRNAs indicating these effects were due to both treatments(siGli+ribavirin) and not effects of Gli-1 alone. Western blot analysisconfirmed overexpression or knockdown of Gli-1. Further, eIF4E levelswere not altered in these conditions.

A variety of sonic hedgehog pathway inhibitors have been developed andsuccessfully used for targeting Smoothed, the extracellular receptor forsonic hedgehog signaling. One of these, GDC-0449, has been used inclinical trials for a variety of malignancies. To determine whether typeII ribavirin resistance was sensitive to pharmacological intervention,the ability of GDC-0449 to revert resistance in F10R cells was assessed(FIG. 7.). Parental FaDu cells or F10R cells were pretreated with 3 nMGDC-0449 or mock treated and after 48 hours, were mock treated ortreated with 10 μM ribavirin. In parental cells, 10 μM ribavirin for 96hours led to a decrease in cell number by about 50% whereas 10 μMribavirin did not effect growth of F10R cells relative to untreated F10Rcells. Strikingly, the combination of ribavirin and GDC-0449 cells ledto a 40% drop in cell number relative to the other treatments oruntreated controls in F10R cells. Although this is not the full 50% dropobserved in ribavirin treated parental cells, it clearly indicates thatthe addition of GDC-0449 to ribavirin resensitizes cells to ribavirin.This parallels the Gli-1 knockdown experiment described above. Incontrast in parental cells, this combination did not reduce cell numberbelow ribavirin only treated cells. Further, GDC-0449 alone led to a 25%reduction in growth in parental cells whereas GDC-0449 alone did notaffect F10R cells. Finally, GDC-0449 had no effect on the growth of type1 resistant F100R cells, which do not have elevated Gli-1. Takentogether with above findings, these studies strongly suggest that typeII ribavirin resistance can, at least in large part, be reverted byinhibition of the sonic hedgehog pathway. Further, the F10R cells havedeveloped a co-dependency on Gli-1 and eIF4E pathways as observed by theneed for both ribavirin and GDC-0449 to reduce growth. According to thepresent invention, Gli-1 knockdown did not affect the growth of F10Rcells alone, but did in combination with ribavirin.

It was observed that the phase II drug metabolism pathway in the form ofthe UGT glucuronidation pathway was highly elevated in F10R cells,strongly suggesting that ribavirin is being glucuronidated in thesecells as a novel form of resistance. The small molecule inhibitor of thesonic hedgehog pathway, GDC-0049 revert ribavirin resistance.Strengthening the tie between Gli-1 and resistance is the fact thatgenetic knockdown of Gli-1 also revert resistance and Gli-1overexpression in ribavirin sensitive cells imparts resistance.

Gli-1 mRNA levels were monitored in patients before, during, and afterresponse in patients receiving ribavirin monotherapy. To date, thenon-responding patients before treatment had over 20 fold elevatedribavirin levels. These levels were not reduced during ribavirintreatment, indeed for one case that there is available data, the Gli-1levels nearly doubled. For responding patients, in all cases Gli-1 mRNAlevels were higher at resistance than during response. In some cases,ribavirin levels started high in the before treatment sample and thenwere reduced (for instance, patient 17 had 20× Gli-mRNA beforetreatment, 5.8 during blast response and 26× at relapse), whereas inother cases, Gli-1 mRNA levels started low and then became elevated atrelapse (for instance, patient 10 had 1.3× Gli-1 mRNA before treatmentrelative to normal, 1.6× during response and 3.6× at relapse).

Ribavirin Resistance

Alterations in the Proteome and Gene Expression of Resistant Cell Linesas a Function of Ribavirin Treatment

Deep sequencing in 10R cells during ribavirin treatment was carried outin order to monitor inducible changes and compare these to changes inparental cell lines. All deep sequencing in resistant cells is done inthe absence of ribavirin. Addition of ribavirin did not alter the ENT1,ADK or Gli-1 levels.

Approved drugs were studied to ascertain modalities to overcomeresistance, or the appearance of new sensitivities that occur upon theonset of resistance as observed for other drugs. It was also studiedwhether acquired resistance was reversible. After 6 months of growingresistant cells in the absence of ribavirin, these cells retained theirresistance suggesting genetic changes underlie this.

The molecular underpinnings of primary and acquired resistance toribavirin were assessed using two strategies:

-   -   to generate resistant cell lines; and    -   to analyze patient specimens for the expression of genes        involved in ribavirin metabolism.

It has been noted that patients that only achieved PD in either trial,did not have a molecular response. Thus, the possibility that patientswith no molecular response had issues with ribavirin uptake ormetabolism was considered, or resistance arose due to elevated Gli-1.

The Applicant studied multiple ribavirin resistant cell lines. Resistantcell lines were generated by either slowly increasing ribavirinconcentrations or by growing at constant ribavirin concentrations atclinically achievable doses. During the study, mutations in the eIF4Egene product were not observed in cell lines and are in the process ofsequencing the eIF4E gene product in patient samples. Reduced/elevatedthe eIF4E gene product levels as a function of resistance in cell lineswere not observed even after culturing in ribavirin for over 200 days.In all of studied resistant cell lines, a loss of ribavirin mediatedgrowth inhibition was observed and a loss of response of eIF4E targetsto ribavirin. Consistently, the eIF4E gene product can no longerimmunoprecipitate ³H ribavirin in resistant cells in contrast tocontrols.

Both a candidate approach and deep sequencing were used to characterizeribavirin resistant cell lines. In two of the cell lines grown in 100 μMor 20 μM ribavirin (100R and 20R), cells had a severely impaired netuptake of ³H-ribavirin. Adenosine kinase (ADK) RNA levels were lowered40-fold in 100R cells and both ADK and ENT1 were similarly reduced in20R cells. ADK phosphorylates ribavirin to yield ribavirin monophosphate(RMP) as the first step to form its active metabolite RTP. Ribavirin isonly exported via the ENT1 nucleoside transporter when it is notphosphorylated. Thus, reduction in ADK increases the pool of exportableribavirin. The applicant noted that 10R cells showed no difference inADK or ENT1 relative to parental cells nor did they have impairedribavirin net uptake-3H ribavirin IP. Growth studies and monitoring theeIF4E gene product targets indicates that the 10R cells are resistant tothe effects of ribavirin.

Gene expression changes using deep sequencing was assessed. Performedstudies reveal a drastic increase in the RNA levels of Gli-1 (17×) andPDEA2 (22×) both of which have been linked to the establishment ofresistance in F10R cells. Thus, F10R cells may have undergonesubstantial genetic rewiring and likely have lost their addiction toeIF4E. Applicant has noted in light of the studies performed thatribavirin resistance can arise due to multiple mechanisms. Whether thesemechanisms were relevant or not in patients from the monotherapy trialwas assessed. Patient 11 (CR), showed a 20-fold reduction in ENT1 and a5-fold reduction in ADK RNA levels at relapse relative to duringresponse (after 9 cycles of treatment) (See FIG. 2). Gli-1 levelschanged (see table).

CT is the number of 28 day cycles of ribavirin, normal is derived fromnormal CD34+ cells from the bone marrow. Results from RT-qPCR arenormalised against multiple genes to ensure no changes in the normalisedoccurred as described. There was no more before treatment RNA availablefor patient 11, but he was responding at cycles 3 and 4 and clinical andmolecular relapse occurred at cycle 9.

In a PD, 5-fold lower ADK levels prior to therapy were observed comparedto normal controls but had normal ENT1 levels. Patient 10 (BR, AMLsecondary to breast cancer) had a 3-fold reduction in ENT1 levels andconcomitant 17-fold increase in MDR1 at relapse at four cycles. In otherpatients, none of these factors explained primary resistance or theonset of acquired resistance underlying the importance of studies inF10R cells and more patient specimen analysis. Clearly, in theheterogeneous background of these patients, resistance arises throughmultiple means.

There was no competition observed for the uptake of ribavirin and Ara-C®in patient specimens or cell lines. Both Ara-C® and ribavirin are takenup by nearly all cell types using the ENT1 transporter and the oralabsorption issues have been identified in phase I trial and haveexamined ribavirin and Ara-C® uptake. Using ribavirin and ³H-Ara-C®, nocompetition in terms of uptake for either drug was observed atclinically relevant concentrations (and higher) in patient specimens orcell lines.

Glucuronidation Enzymes are Elevated in Type II Resistant Cells

A deep sequencing study also revealed a striking change in the enzymesin the glucuronidation pathway in F10R cells. The UDPglucuronosyltransferase type 1A (UGT1A) family of enzymes plays keyroles in phase II detoxification and drug metabolism. Although theseactivities were initially considered limited to the liver, most celltypes are now known to have UGT activity. Surprisingly, there was the 14fold depletion in the RNA levels of nearly all the UGT1A family ofenzymes including A1, A3, A4, A5, A6, A7, A8, A9 and A10 (padj valuesfrom 5×10-8 to 1×10-9). This depletion was confirmed by qRT-PCR in F10Rcells. Note that UGT1A2 RNA was not detected in any of the cell linesexamined, including parental cells. Importantly, there was no changeobserved in the UGT2 class of these enzymes. The UGT1A enzymes aretranscribed from a common gene locus through an exon sharing mechanismand thus have a common carboxy terminus, and all have approximately thesame molecular weight. Thus, an antibody that recognizes the UGT1Afamily was used to assess protein levels (FIG. 8). In direct contrast tothe RNA levels, protein levels of the UGT1A family were highly elevated(3-4 fold) in F10R cells relative to parental cell lines or F100Rs. Thisis evidence of a complex regulatory mechanism governing the UGT pathwayin F10R cells.

Whether there was a link between Gli-1 elevation and increased proteinexpression of UGT was examined. Parental FaDu cells overexpressing GLi-1had higher UGT levels relative to vector controls. RNAi mediatedknockdown of Gli-1 led to substantial reduction (over 10 fold) in UGT1Aprotein levels compared to the knockdown of luciferase RNA and untreatedcontrols. Consistent with these findings, GDC-0449 treatment led to asimilar decrease in UGT1A levels. Taken together, these results providea strong link between Gli-1 levels, and protein expression of UGT.Controls confirmed that Gli-1 overexpression or reduction in knockdowncells. Further, none of these treatments modulated eIF4E levels. UGT1Aprotein expression strongly correlates with its enzyme activity. Thus,it was hypothesized that glucuronidation activity, at least for somesubstrates, was elevated in F10R cells relative to parental controls.

Glucuronidation of Ribavirin is Elevated in Resistant Cells and ImpairsInteraction with EIF4E

The UGT1A family catalyzes the addition of UDP-glucuronic acid (UDPGA)to a myriad of substrates including triazoles, the same chemical classas ribavirin. Importantly, different UGT enzymes have different targetspecificities and thus substrates can be glucuronidated in a tissue andcell type in a specific manner. Whether there was differentialglucuronidation of ribavirin in FaDu cells versus F10R cells wasmonitored. Using cell/microsomal lysates, ¹⁴C-UDPGA and ribaviringlucoronidation was monitored using thin layer chromatorgraphy. Theconverse experiment with unlabelled UDPGA and ¹⁴C-ribavirin was alsocarried out. As a positive control, the glucuronidation ofbenzo(a)pyrene, a common target of this pathway that is one of the maincarcinogens in cigarette smoke was monitored.

It was hypothesized that glucuronidation of ribavirin impairs itsassociation with eIF4E. The ability of eIF4E to immunopreciptate with¹⁴C-ribavirin in conditions that varied in Gli-1 and UGT activity wasmonitored to determine if reversion of resistance correlated withre-association of ribavirin with eIF4E. Cells were treated for 24 hourswith ¹⁴C-ribavirin followed by cross-linking and immunoprecipitation andevaluated relative to IgG controls. This demonstrated that in F10Rcells, eIF4E did not immunoprecipitate with ribavirin whereas it did inparental cells.

GDC-0449 treatment or Gli-1 knockdown in F10R cells led to an increasein ribavirin binding to eIF4E, which correlated with lowering of UGTlevels. In parental cells overexpressing Gli-1, there was a loss inassociation of ribavirin to eIF4E. This indicates a correlation betweenlower Gli-1 activity, lower UGT levels, and the ability of eIF4E toassociate with unmodified ribavirin and resistance.

GLI-1 Elevation in Clinical Samples from the AML Ribavirin MonotherapyTrial

The molecular features identified in present studies were examined todetermine whether they were relevant to specimens from patients thatreceived ribavirin monotherapy in the multi-centre phase II AML trial.For responding patients, all available specimens before treatment weremonitored, during clinical response, upon the onset of relapse and innon-responders when possible. A total of ten specimens were examinedincluding those from patients that enrolled in the monotherapy trial.Results from the trial, when closed, were 1 CR, 2 PR, 3 BR, 6 SD, 3 PDwith 15 evaluable patients (Clinicaltrials.gov). Of these, samples from10 patients were available for evaluation of molecular response. Theresults from the testing carried out suggest that type II resistance isobserved in many of these patients. Gli-1 mRNA levels were monitoredusing qRT-PCR. Of the 6 responding patients who had specimens atrelapse, all showed elevated Gli-1 mRNA levels, up to 26 fold, uponresistance relative to normal controls or their levels during response.Some patients have elevated Gli-1 prior to ribavirin monotherapy, whichfalls during response and reemerges at loss of response. For the twopatients examined that did not respond (PD), both had highly elevatedGli-1 levels prior to treatment (19 and 800 fold), which was not loweredafter 28 days of ribavirin. Taken together, it appears that elevatedGli-1 is associated with primary and acquired resistance in patients inthe ribavirin monotherapy trial. (See above table).

In terms of type I resistance, one non-responder (patient 9) wasobserved to have five times lower ADK levels than specimens from healthyvolunteers prior to treatment. In the patient that achieved a CR(patient 11), it was observed that during response ENT1 and ADK RNAlevels fell 15 and 5 fold, respectively, upon relapse relative toresponse. Thus, there is evidence that type I resistance plays a role inpatients. However, the remaining patients did not have lower ADK RNAlevels or marked alterations in ENT (at the RNA level). These findingssuggest that type I resistance can also contribute to primary andacquired resistance in at least some patients. Further, patient 11 hadboth lower ADK levels and highly elevated Gli-1 levels, suggesting thatthese forms of resistance can co-emerge.

In contrast to type I resistance, type II is characterized by no defectin ribavirin uptake but a loss of the eIF4E-ribavirin interactioncorrelating with the elevation of Gli-1 mRNA and protein levels.Strikingly, overexpression of Gli-1 alone was sufficient to makeparental cells resistant to ribavirin and was sufficient to abrogate theeIF4E-ribavirin interaction. In contrast, pharmacological inhibitionusing GDC-0049 or genetic knockdown of Gli-1 revert ribavirin resistancein the F10R cells and this correlates with a re-establishment of theeIF4E-ribavirin interaction. Importantly, primary and acquiredresistance in the ten patient specimens examined indicated that Gli-1levels were high prior to therapy and/or became elevated at the onset ofresistance suggesting that targeting Gli-1 with GDC0049, or relateddrugs, in patients could help overcome primary resistance and perhaps,prolong clinical responses.

Studies with chemical carcinogens link lowered UGT1A levels withincreased carcinogenesis because of the loss of this importantdetoxification pathway. Gilbert's and Crigler-Najjar' syndromes, geneticdiseases characterized by mutation of UGT1A1, lead to decreasedglucuronidation of bilirubin, and varying severity in associatedtoxicity. The inventors of the present invention have determined thatincreased UGT1A activity shows a correlation to drug resistance. In thisway, the cancer cell can subvert normal detoxification processes inorder to modify the drug and become resistant. The inventors of thepresent invention have identified a drug, GDC-0449, that can modulateglucuronidation, albeit indirectly.

Drug resistance is usually considered to occur by one of two mechanisms.First, as observed in type I resistance, there is either a loss ormutation of relevant drug transporters or in the enzymes that convertpro-drugs to their active forms. Alternatively there ismutation/modification of the protein target/pathway. However, mutationin the eIF4E gene in studied cell lines was not observed. In contrast,Gleevec® or ATRA treatment can lead to mutations in Bcr-AbI and PML-RARAfusion proteins, respectively, which drive resistance. While neitherBcr-AbI nor PML-RARA are required for survival of normal cells, eIF4E isessential where in yeast, its knockout is lethal. Thus, stochasticmechanisms that drive resistance via mutation in the cap-binding site ofeIF4E likely end with slow growing or dying cell populations and thus,are easily competed out by other forms of resistant cells. In this way,resistant mechanisms targeting proteins that are required for cellularsurvival may be distinct from those targeting proteins that aredispensable for normal cellular function. In these situations,resistance can be driven by chemical modification of the drug ratherthan only by mutation or modification of the target protein.

Deep sequencing reveals that cells have also become more malignant inthe face of ribavirin resistance, with levels of eIF4E targets such asVEGF becoming elevated even upon ribavirin treatment.

The invention claimed is:
 1. A pharmaceutical composition comprising apharmaceutically acceptable carrier and therapeutically effectiveamounts of ribavirin and GDC-0449 in the form of a combined preparation.2. The pharmaceutical composition according to claim 1, furthercomprising a therapeutically effective amounts of a chemotherapeuticagent.
 3. The pharmaceutical composition according to claim 2, whereinthe chemotherapeutic agent is cytarabine.
 4. The pharmaceuticalcomposition according to claim 1, wherein the therapeutically effectiveamounts of ribavirin is between about 1000 and about 4400 mg per day. 5.The pharmaceutical composition according to claim 3, wherein thepharmaceutical composition comprises a low dose of cytarabine.
 6. Thepharmaceutical composition according to claim 5, wherein the low dose ofcytarabine ranges from about 10 mg per day to about 20 mg twice a day.7. The pharmaceutical composition according to claim 5, wherein the lowdose of cytarabine ranges from about 5 μM to about 15 μM per day.
 8. Thepharmaceutical composition according to claim 1, wherein saidcomposition is suitable for inhalation, ocular administration, nasalinstillation, parenteral administration, dermal administration,transdermal administration, buccal administration, rectaladministration, sublingual administration, perilingual administration,nasal administration, topical administration or oral administration. 9.A method for the treatment of leukemia comprising: administering atherapeutically effective amount of ribavirin and a therapeuticallyeffective amount of GDC-0449.
 10. The method according to claim 9,further comprising administering a therapeutically effective amounts ofa chemotherapeutic agent.
 11. The method according to claim 10, whereinthe chemotherapeutic agent is cytarabine.
 12. The method according toclaim 9, wherein ribavirin and GDC-0449 are separately, sequentially orsimultaneously co-administered.
 13. The method according to claim 11,wherein the therapeutically effective amounts of ribavirin and GDC-0449are separately, sequentially or simultaneously co-administered prior toadministration of the cytarabine.
 14. The method according to claim 13,wherein the method comprises administering ribavirin and GDC-0449 in asingle unit dosage form.
 15. The method according to claim 9, whereinthe ribavirin is administered in an amount between 1000 and 4400 mg perday.
 16. The method according to claim 11, wherein the cytarabine isadministered in a low dose.
 17. The method according to claim 16,wherein the low dose ranges from about 10 mg per day to about 20 mgtwice a day.
 18. The method according to claim 16, wherein the low doseof cytarabine ranges from about 5 μM to about 15 μM per day.
 19. Themethod according to claim 10, wherein the chemotherapeutic agent isadministered in an amount up to 100 mg/m² per day, for up to 7 daysevery 4 weeks.
 20. The method according to claim 12, wherein thetherapeutically effective amount of GDC-0449 ranges from about 3 μM fortwo days prior to start of administration of the ribavirin.
 21. Themethod according to claim 9, wherein the leukemia is selected from thegroup consisting of: acute myeloid leukemia, acute myelocytic leukemia,acute myeloblastic leukemia, acute lymphoblastic leukemia, acutepromyelocytic leukemia, acute myelomonocytic leukemia, acute monocyticleukemia, acute erythroleukemia, chronic leukemia, chronic myelocyticleukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia,and myelodysplastic syndromes.
 22. The method according to claim 21,wherein the acute myeloid leukemia is selected from the group consistingof: acute myeloid leukemia M4, acute myeloid leukemia M5 and AMLsubtypes characterized by atypical elevation of the e1F4E gene product.23. The method according to claim 14, wherein said composition issuitable for inhalation, ocular administration, nasal instillation,parenteral administration, dermal administration, transdermaladministration, buccal administration, rectal administration, sublingualadministration, perilingual administration, nasal administration,topical administration or oral administration.
 24. The method accordingto claim 9, comprising administering a therapeutically effective amountof GDC-0449 in the presence of a therapeutically effective amount ofribavirin, wherein the resistance previously developed in patientsduring anti-leukemia treatment is minimized by the reduction of UGT1Aproteins levels and inhibition of the Gli-1 overexpression.
 25. Themethod according to claim 9, wherein the ribavirin and GDC-0449 areadministered orally.
 26. The method according to claim 14, wherein saidsingle unit dosage form is administered orally.
 27. A combinationcomprising a first pharmaceutical composition including apharmaceutically acceptable carrier and a therapeutically effectiveamount of ribavirin and a second pharmaceutical composition including apharmaceutically acceptable carrier and a therapeutically effectiveamount of GDC-0449.